Historically, the detection and identification of bacteria, mycobacteria, yeasts, and molds have relied primarily on their morphologic and phenotypic properties. This approach is imprecise and slow for many clinically significant microbes. We have explored alternative methods, such as targeted sequencing and mass spectrometry, for the detection and identification of selected organisms. Studies in the current fiscal year have focused on improving protocols and speed of MALDI-TOF (matrix-assisted laser desorption-ionization time of flight mass spectrometry) for the identification of nocardia, mycobacteria, and molds and rapid genomic assays for identification, typing and detection of antibiotic resistance in M. abscessus group. MALDI-TOF MS provides a reproducible spectral pattern based on the mass/charge (m/z) ratio of ionized proteins. We have recently developed and validated a simple and rapid MALDI-TOF MS extraction protocol for mycobacteria, nocardia and filamentous molds which allows faster turn around time. A multi center evaluations of the performance of the NIH mold database is ongoing. Finally, we are exploring the use of proteomics and in particular LC-MS for direct detection of microbial antigens in clinical samples and our project were granted a Bench to Bedside (BtB) award. Our findings support the validity of this approach for detection of circulating antigens of Mycobacterium tuberculosis and is being currently extended to other mycobacterial species as well as other bacterial pathogens.